Figure 5. Simultaneous generation of three distinct allelic exchange events.

(A) Schematic of the approach used to conditionally disrupt pfpkg and create three distinct knockout parasite populations expressing either mTagBFP2 (ΔPKG_BFP2), eGFP (ΔPKG_GFP), or mCherry (ΔPKG_mCherry). Positions of lox sites are indicated with coloured arrowheads (yellow, loxN; green, lox2272; purple, lox71; brown, lox66). Positions of oligonucleotide primers used for diagnostic PCR are indicated (coloured arrows). (B) Confirmation by diagnostic PCR of the three recombination events (RE1, RE2, and RE3, respectively) in RAP-treated pkg:3cKO parasites. Coloured arrows represent identify of the primers used. (C) Growth assay of the pfpkg_3lox and pkg:3cKO parasite lines after mock-treatment (DMSO) or treatment with RAP. Parasitaemia was measured by flow cytometry. Error bars ± SD (n = 6). (D) Fluorescent microcopy images of live RAP-treated pkg:3cKO schizonts from the end of cycle 0, confirming the presence of all three fluorescent populations. Scale bars, 10 μΜ.